CN101655496A - Test strip for quickly detecting ureaplasma urealyticum antibodies in saliva - Google Patents
Test strip for quickly detecting ureaplasma urealyticum antibodies in saliva Download PDFInfo
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- CN101655496A CN101655496A CN200910152611A CN200910152611A CN101655496A CN 101655496 A CN101655496 A CN 101655496A CN 200910152611 A CN200910152611 A CN 200910152611A CN 200910152611 A CN200910152611 A CN 200910152611A CN 101655496 A CN101655496 A CN 101655496A
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Abstract
The invention discloses a test strip for quickly detecting ureaplasma urealyticum antibodies in saliva. The test strip comprises a bottom plate on which a sample pad, a collaurum pad, a nitrocellulosefilm and a sample sucking pad are sequentially pasted, wherein the collaurum pad is attached with mouse anti-human IgG antibodies which are labeled by collaurum and can combine with the specificity of the ureaplasma urealyticum antibodies; and a detection line consisting of ureaplasma urealyticum antigens and a quality control line consisting of sheep anti-mouse secondary antibodies which can combine with the specificity of the mouse anti-human IgG antibodies are coated on the nitrocellulose film. The test strip adopts a collaurum labeling technique to detect the ureaplasma urealyticum antibodies in saliva so as to judge whether a testee infects ureaplasma urealyticum or not. The invention has the advantages of simple operation, high reaction speed and sensibility, strong specificity, suitability for on-site detection and self detection, economy, practicability, and the like.
Description
Technical field
The present invention relates to technical field of biological, relate in particular to a kind of test strip for quickly detecting ureaplasma urealyticum antibodies in saliva.
Background technology
Ureaplasma urealyticum (M.urealyticum) is unique in a Ureaplasma kind, because of growth needs urea is gained the name.Bacterium colony is small, and diameter only has 15～25um, must observe under low-power microscope, and old friend claim T strain (tiny strain).There is coarse particles on the bacterium colony surface, can change into typical poached egg sample bacterium colony under appropraite condition.Growth needs cholesterol and urea, decomposing urea are its metabolic characteristics, produce ammonia nitrogen, and medium pH is risen, and patient's urine often has the urine smell flavor.
The healthy relation of Ureaplasma urealyticum and female reproduction is the closest, and Ureaplasma urealyticum can cause urogenital infections, and is considered to be only second in the non gonococcal urethritis the important pathogen of Chlamydia (accounting for 50%).Owing to have Ureaplasma urealyticum in 80% pregnant woman's the genital tract, therefore can cause premature labor, stillborn foetus, or when childbirth, infect the neonate by the placental infection fetus, cause respiratory tract infection.In addition, Ureaplasma urealyticum also can cause infertility.
The life of the main trafficability characteristic of ureaplasma urealyticum infection is propagated, and is more common in youth vigorous period, be more common in unclean sexual intercourse especially after.When urogenital tract is inflamed, Ureaplasma urealyticum was easily invaded from damaged mouthful when mucomembranous surface was impaired, caused urogenital infections.Behind the ureaplasma urealyticum infection, therefore the most non-evident sympton of patient, is difficult to be perceiveed by the patient, and also Yi Zaocheng doctor fails to pinpoint a disease in diagnosis.Ureaplasma urealyticum can be invaded urea road, uterine neck and greater vestibular gland, causes urea road inflammation, cervicitis and bartholinitis; During up infection, can cause endometritis, pelvic infecton, salpingitis, especially salpingitis is seen more.The female sex organ pathologic that ureaplasma urealyticum infection causes changes, and is infertile major reason.
Data at home and abroad prompting, Ureaplasma urealyticum is cultivated positive rate up to more than 50% in the cervical mucus of Infertile Couples, the seminal fluid, this shows, ureaplasma urealyticum infection and infertility correlationship arranged.It is miscarriage that ureaplasma urealyticum infection causes bad Another reason, has the people to check out that the positive rate of Ureaplasma urealyticum is up to more than 40% from the tissue of miscarriage.Therefore, to the miscarriage of unknown cause, Aborter especially repeatedly should consider to have the possibility of ureaplasma urealyticum infection.The fallopian tubal inflammatory adhesion of the partial obstruction that ureaplasma urealyticum infection causes can make luminal stenosis, and is logical and not smooth, and the major reason of ectopic pregnancy still takes place.
At present, detecting the most frequently used method of above-mentioned infectious disease is immune detection, and the method comprises two kinds of traditional enzyme linked immunosorbent assay (ELISA) and the recent colloidal gold methods that rises based on the specific recognition of Ag-Ab.Colloidal gold method is divided into immunochromatography and immunity percolation dual mode, and is wherein convenient, fast with immunochromatographic method.
The immunochromatography colloidal gold technique is novel diagnostic techniques, obtained using comparatively widely, ultimate principle is as follows: utilize a kind of antigen of colloid gold label or antibody, bag is matched antigen or antibody accordingly on the nitrocellulose filter of test strips, during detection when containing corresponding specific antibody or antigen in the sample, the part formation compound that combines in colloid gold label particle and the sample, chromatography on nitrocellulose filter then, coated again antigen or antibody capture, form macroscopic detection line (T line), have or not the judgement of realization the result by detection line.But existing test strips is a sample with blood mostly, is not suitable for on-the-spot the detection and self check, and it is higher to detect cost.
Summary of the invention
The invention provides a kind of test strip for quickly detecting ureaplasma urealyticum antibodies in saliva, this test strips can solve existing test strips and be not suitable for on-the-spot the detection with saliva as sample, detects the cost problem of higher.
A kind of test strip for quickly detecting ureaplasma urealyticum antibodies in saliva, comprise base plate, be pasted with sample pad, collaurum pad, nitrocellulose filter on the base plate successively and inhale the sample pad, be attached with the mouse-anti human IgG antibody that can combine of colloid gold label on the described collaurum pad, be coated with the detection line that constitutes by Ureaplasma urealyticum antigen on the described nitrocellulose filter and by the nature controlling line that can constitute with sheep anti mouse two antiantibodys that described mouse-anti human IgG antibody specificity combines with the ureaplasma urealyticum antibodies specificity.
Whether described ureaplasma urealyticum antibodies is medically to detect the people to infect and separate the chlamydial label of urea, and this label is present in the blood of human body in a large number, and a small amount of existence is also arranged in people's saliva.Described Ureaplasma urealyticum antigen can combine with the ureaplasma urealyticum antibodies specificity, is people's recombinant antigen.Described mouse-anti human IgG antibody, Ureaplasma urealyticum antigen and sheep anti mouse two antiantibodys can prepare by commercially available or existing method.
Be attached with 0.15～0.25 μ g/cm on the described collaurum pad
2Described mouse-anti human IgG antibody can satisfy test strips sensitivity requirement.
Described collaurum pad preparation method is as follows:
Get the colloidal gold solution that the colloid diameter is 15～35nm, regulate pH value to 8.0～8.2, in every 100ml colloidal gold solution, add the described mouse-anti human IgG antibody of 0.55～1.2mg, stirring the back seals with bovine serum albumin, the centrifugal supernatant that goes, the colloidal gold solution that adds equal volume redissolves, and every ml soln evenly is layered on 40cm
2On the glass fibre membrane, make the collaurum pad after the drying.
The content of the Ureaplasma urealyticum antigen described in the described detection line is 0.06～1.0 μ g/cm; Sheep anti mouse two antiantibody content described in the described nature controlling line are 0.06～1.0 μ g/cm, can satisfy the requirement of test strips detection sensitivity.
Described detection line method for coating is as follows:
Described Ureaplasma urealyticum antigen is dissolved in to make concentration in the phosphate buffer solution be 0.06～0.9mg/ml solution, with the end line of the consumption of 1～1.5 μ l/cm, promptly gets detection line after the drying at nitrocellulose filter with this solution;
Described nature controlling line method for coating is as follows:
Described sheep anti mouse two antiantibodys are dissolved in to make concentration in the phosphate buffer solution be 0.06～0.9mg/ml solution, with the other end line of the consumption of 1～1.5 μ l/cm, promptly get nature controlling line after the drying at nitrocellulose filter with this solution.
The present invention also provides the detection method of above-mentioned test strip for quickly detecting ureaplasma urealyticum antibodies in saliva, may further comprise the steps:
Get people's saliva, with the phosphate buffer solution dilution, the saliva of getting after 50～100 μ l dilute drops on the sample pad, according to the color status of detection line (T line) and nature controlling line (C line), judges testing result.
All occur as C, T line, judge that sample is positive; The C line occurs, and the T line does not occur, and judges that sample is negative; C, T line do not occur or the appearance of T line does not appear in the C line, judge that all test paper is invalid.
Test strips of the present invention adopts colloidal gold-labeled method, detect the ureaplasma urealyticum antibodies in the saliva, thereby judge whether to infect Ureaplasma urealyticum, have simple to operate, reaction fast, high, the high specificity of susceptibility, be fit to on-the-spot detection and self check, advantage such as economical and practical.
Embodiment
Embodiment 1
The preparation of collaurum pad
Prepare the colloidal gold solution of diameter 15nm with gold chloride-trisodium citrate reduction method, get the 100ml colloidal gold solution in beaker after preparation is finished, use 0.1M K
2CO
3Transfer to pH8.0, add the mouse-anti human IgG antibody (Shanghai Linc-Bio Science Co., Ltd.) that 0.55mg can combine with the ureaplasma urealyticum antibodies specificity, stirring at room 1 hour, added percentage by weight and be 5% bovine serum albumin(BSA) (BSA) sealing 1 hour, 12000rpm, 4 ℃ are centrifugal 30 minutes, abandon supernatant, redissolve to 100ml, press 1ml solution shop 40cm with colloidal gold solution
2Ratio, solution is layered on the glass fibre membrane equably, 37 ℃ of dryings 30 minutes are made the collaurum pad.
The bag quilt of nitrocellulose filter
The Ureaplasma urealyticum antigen (Shanghai Linc-Bio Science Co., Ltd.) that can combine with the ureaplasma urealyticum antibodies specificity is dissolved in the solution that 0.01M, pH8.0 phosphate buffer (PBS) are mixed with 0.06mg/ml, rules with the parameter of 1.5 μ l/cm at nitrocellulose filter one end with spray film instrument.
Can be dissolved in 0.01M, pH8.0 phosphate buffer (PBS) with sheep anti mouse two antiantibodys (Shanghai Linc-Bio Science Co., Ltd.) that above-mentioned mouse-anti human IgG antibody specificity combines and be mixed with the solution of 0.06mg/ml, rule with the parameter of 1 μ l/cm at the nitrocellulose filter other end with spray film instrument.After the line at room temperature dry 8 hours, obtain detection line and nature controlling line respectively.
Detect the assembling of test paper
In hothouse, 20～25 ℃ of temperature, humidity is less than 30%, sample pad (glass fibre membrane), collaurum pad, nitrocellulose filter and suction sample pad (suction test paper) are sticked on the base plate, wherein nitrocellulose filter is positioned at the base plate middle part, nitrocellulose filter is coated with an end of T line and 1/3 overlap joint of collaurum pad, is coated with an end and 1/10 overlap joint of inhaling the sample pad of C line, 1/5 overlap joint of sample pad and collaurum pad.Be cut into the test strips that width is 2.5mm at last, make test card in the plastic clip of also test strips can being packed into.
Embodiment 2
The preparation of collaurum pad
Prepare the colloidal gold solution of diameter 35nm with gold chloride-trisodium citrate reduction method, get the 100ml colloidal gold solution in beaker after preparation is finished, use 0.1M K
2CO
3Transfer to pH8.2, add the mouse-anti human IgG antibody (Shanghai Linc-Bio Science Co., Ltd.) that 1.2mg can combine with the ureaplasma urealyticum antibodies specificity, stirring at room 1 hour, added percentage by weight and be 5% bovine serum albumin(BSA) (BSA) sealing 1 hour, 12000rpm, 4 ℃ are centrifugal 30 minutes, abandon supernatant, redissolve to 100ml, press 1ml solution shop 40cm with colloidal gold solution
2Ratio, solution is layered on the glass fibre membrane equably, 37 ℃ of dryings 30 minutes are made the collaurum pad.
The bag quilt of nitrocellulose filter
The Ureaplasma urealyticum antigen (Shanghai Linc-Bio Science Co., Ltd.) that can combine with the ureaplasma urealyticum antibodies specificity is dissolved in the solution that 0.01M, pH8.0 phosphate buffer (PBS) are mixed with 0.9mg/ml, rules with the parameter of 1.5 μ l/cm at nitrocellulose filter one end with spray film instrument.
Can be dissolved in 0.01M, pH8.0 phosphate buffer (PBS) with sheep anti mouse two antiantibodys (Shanghai Linc-Bio Science Co., Ltd.) that above-mentioned mouse-anti human IgG antibody specificity combines and be mixed with the solution of 0.9mg/ml, rule with the parameter of 1.5 μ l/cm at the nitrocellulose filter other end with spray film instrument.After the line at room temperature dry 8 hours, obtain detection line and nature controlling line respectively.
Detect the assembling of test paper
In hothouse, 20～25 ℃ of temperature, humidity is less than 30%, sample pad (glass fibre membrane), collaurum pad, nitrocellulose filter and suction sample pad (suction test paper) are sticked on the base plate, wherein nitrocellulose filter is positioned at the base plate middle part, nitrocellulose filter is coated with an end of T line and 1/3 overlap joint of collaurum pad, is coated with an end and 1/10 overlap joint of inhaling the sample pad of C line, 1/5 overlap joint of sample pad and collaurum pad.Be cut into the test strips that width is 2.5mm at last, make test card in the plastic clip of also test strips can being packed into.
The clinical sample test
People to be checked is gathering saliva sample fasting in preceding 30 minutes, get every people's saliva 0.5ml to be checked in dixie cup, get in two phosphate buffer solutions that are added drop-wise to concentration 0.02M, pH8.0 with suction pipe and to dilute, the saliva 100 μ l that get dilution are added in the sample pad, naked-eye observation 30 minutes, the color status of record T line and C line.Simultaneously saliva sample is detected with ureaplasma urealyticum antibodies ELISA kit, if both test result are inconsistent, detect with other two kinds of ureaplasma urealyticum antibodies ELISA kits again, if two kinds or above ELISA reagent are positive, result of determination is positive, two kinds or above ELISA reagent are negative, and result of determination is negative.
Collect 163 parts of saliva samples, wherein the ELISA kit detects 43 parts of positive, and test strips of the present invention (embodiment 1) detects 44 parts of positive, and concrete outcome is as shown in table 1 below:
Table 1 test strips of the present invention is to the testing result of clinical sample ureaplasma urealyticum antibodies
Sensitivity=43/43=100%; Specificity=119/120=99.2%.
Claims (6)
1, a kind of test strip for quickly detecting ureaplasma urealyticum antibodies in saliva, comprise base plate, be pasted with sample pad, collaurum pad, nitrocellulose filter on the base plate successively and inhale the sample pad, it is characterized in that: be attached with the mouse-anti human IgG antibody that can combine of colloid gold label on the described collaurum pad, be coated with the detection line that constitutes by Ureaplasma urealyticum antigen on the described nitrocellulose filter and by the nature controlling line that can constitute with sheep anti mouse two antiantibodys that described mouse-anti human IgG antibody specificity combines with the ureaplasma urealyticum antibodies specificity.
2, test strip for quickly detecting ureaplasma urealyticum antibodies in saliva according to claim 1 is characterized in that: be attached with 0.15～0.25 μ g/cm on the described collaurum pad
2The mouse-anti human IgG antibody that can combine with the ureaplasma urealyticum antibodies specificity.
3, test strip for quickly detecting ureaplasma urealyticum antibodies in saliva according to claim 1 and 2 is characterized in that, described collaurum pad preparation method is as follows:
Get the colloidal gold solution that the colloid diameter is 15～35nm, regulate pH value to 8.0～8.2, in every 100ml colloidal gold solution, add the described mouse-anti human IgG antibody of 0.55～1.2mg, stirring the back seals with bovine serum albumin, the centrifugal supernatant that goes, the colloidal gold solution that adds equal volume redissolves, and every ml soln evenly is layered on 40cm
2On the glass fibre membrane, make the collaurum pad after the drying.
4, test strip for quickly detecting ureaplasma urealyticum antibodies in saliva according to claim 1 is characterized in that: the content of the Ureaplasma urealyticum antigen described in the described detection line is 0.06～1.0 μ g/cm; Sheep anti mouse two antiantibody content described in the described nature controlling line are 0.06～1.0 μ g/cm.
According to claim 1 or 4 described test strip for quickly detecting ureaplasma urealyticum antibodies in saliva, it is characterized in that 5, described detection line method for coating is as follows:
Described Ureaplasma urealyticum antigen is dissolved in to make concentration in the phosphate buffer solution be 0.06～0.9mg/ml solution, with the end line of the consumption of 1～1.5 μ l/cm, promptly gets detection line after the drying at nitrocellulose filter with this solution;
Described nature controlling line method for coating is as follows:
Described sheep anti mouse two antiantibodys are dissolved in to make concentration in the phosphate buffer solution be 0.06～0.9mg/ml solution, with the other end line of the consumption of 1～1.5 μ l/cm, promptly get nature controlling line after the drying at nitrocellulose filter with this solution.
6, the detection method of the described test strip for quickly detecting ureaplasma urealyticum antibodies in saliva of a kind of claim 1 may further comprise the steps:
Get people's saliva, with the phosphate buffer solution dilution, the saliva of getting after 50～100 μ l dilute drops on the sample pad, according to the color status of detection line and nature controlling line, judges testing result.
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| Application Number | Priority Date | Filing Date | Title |
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| CN200910152611A CN101655496A (en) | 2009-09-10 | 2009-09-10 | Test strip for quickly detecting ureaplasma urealyticum antibodies in saliva |
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| CN200910152611A CN101655496A (en) | 2009-09-10 | 2009-09-10 | Test strip for quickly detecting ureaplasma urealyticum antibodies in saliva |
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| CN101655496A true CN101655496A (en) | 2010-02-24 |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102183633A (en) * | 2011-02-24 | 2011-09-14 | 南京基蛋生物科技有限公司 | Colloidal gold labeling method |
| CN102445535A (en) * | 2011-11-15 | 2012-05-09 | 中国农业科学院兰州兽医研究所 | Contagious caprine plueropneumonia antibody detection test strips, and preparation method thereof |
| CN104316703A (en) * | 2014-11-05 | 2015-01-28 | 中国农业科学院兰州兽医研究所 | Mycoplasma bovis detection test strip and preparation method thereof |
| CN105968197A (en) * | 2016-05-13 | 2016-09-28 | 湖北工业大学 | Anti-3 type mycoplasma urealytium MB protein antibody and immunochromatography kit applying antibody |
| CN106290884A (en) * | 2015-06-15 | 2017-01-04 | 江苏戴格诺思生物技术有限公司 | A kind of Ureaplasma urealyticum gold mark detection kit and detection method |
| CN111537730A (en) * | 2020-04-22 | 2020-08-14 | 武汉优恩生物科技有限公司 | Test paper for detecting brucella antibody |
-
2009
- 2009-09-10 CN CN200910152611A patent/CN101655496A/en active Pending
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102183633A (en) * | 2011-02-24 | 2011-09-14 | 南京基蛋生物科技有限公司 | Colloidal gold labeling method |
| CN102183633B (en) * | 2011-02-24 | 2013-05-15 | 南京基蛋生物科技有限公司 | Colloidal gold labeling method |
| CN102445535A (en) * | 2011-11-15 | 2012-05-09 | 中国农业科学院兰州兽医研究所 | Contagious caprine plueropneumonia antibody detection test strips, and preparation method thereof |
| CN104316703A (en) * | 2014-11-05 | 2015-01-28 | 中国农业科学院兰州兽医研究所 | Mycoplasma bovis detection test strip and preparation method thereof |
| CN104316703B (en) * | 2014-11-05 | 2016-06-01 | 中国农业科学院兰州兽医研究所 | A kind of Mycoplasma bovis test strip and its preparation method |
| CN106290884A (en) * | 2015-06-15 | 2017-01-04 | 江苏戴格诺思生物技术有限公司 | A kind of Ureaplasma urealyticum gold mark detection kit and detection method |
| CN105968197A (en) * | 2016-05-13 | 2016-09-28 | 湖北工业大学 | Anti-3 type mycoplasma urealytium MB protein antibody and immunochromatography kit applying antibody |
| CN105968197B (en) * | 2016-05-13 | 2019-08-16 | 湖北工业大学 | A kind of anti-3 type Ureaplasma urealyticum MB protein antibodies and the immune chromatography reagent kit using the antibody |
| CN111537730A (en) * | 2020-04-22 | 2020-08-14 | 武汉优恩生物科技有限公司 | Test paper for detecting brucella antibody |
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Application publication date: 20100224 |